Method and apparatus for analyzing vitamin E in lipoproteins

ABSTRACT

A method of analyzing vitamin E components in a lipoproteinby subjecting a lipoprotein-containing sample to ion exchange chromatography to separate the lipoprotein, reacting the separated lipoprotein to a pretreating solution containing an organic solvent and a surfactant to liberate vitamin E components, and then subjecting the liberated vitamin E components to reverse phase chromatography. Also described is a method of judging various pathological conditions such as the pathological conditions of diabetes, the risks of coronary artery diseases, and the pathological conditions of myocardial infarction using levels of vitamin E components in the lipoprotein as an index.

This application is the U.S. national phase of International ApplicationNo. PCT/JP2007/061143, filed 25 May 2007, which designated the U.S. andclaims priority to Japan Application No. 2006-144843, filed May 25,2006, the entire contents of each of which are hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to a method of analyzing vitamin E such asα-tocopherol or γ-tocopherol contained in lipoproteins (high densitylipoprotein/HDL, low density lipoprotein/LDL, intermediate densitylipoprotein/IDL, very low density lipoprotein/VLDL, chylomicron/CM etc.)in a sample, and an analytical apparatus that can be used in such ananalysis. Furthermore, the present invention provides a method ofjudging various pathological conditions such as diabetes and myocardialinfarction, and the risks of coronary artery diseases, using vitamin Esin the lipoprotein as an index.

BACKGROUND ART

As a risk factor for arteriosclerosis, oxidized lipoproteins areattracting a lot of attention and extensive studies are underway, and amajor antioxidant contained in the lipoprotein is vitamin E (mainlyα-tocopherol and γ-tocopherol). Thus, the analysis of vitamin E in eachlipoprotein in the blood is thought to be important in understanding themechanism of arteriosclerosis. Especially, on one of vitamin E,α-tocopherol, abundantly contained in the serum lipoproteins, variousacademic studies have been reported as follows.

(1) Haidari M. et al., Clin. Chem. 47: 1234 (2001);α-tocopherol/cholesterol levels in LDL are reduced in patients withcoronary artery diseases compared to healthy individuals.

(2) Feki M. et al., Clin. Chem. 46: 1401 (2000);α-tocopherol/cholesterol levels in LDL are reduced in patients withcoronary artery diseases compared to healthy individuals.

In the blood of patients with diabetes, it is also believed that activeoxygen (radical) is abundantly generated and thus oxidant stress becomesenhanced, which attacks the cell causing various complications(Beisswenger P J et al., Diabetes 54: 3274 (2005)). This oxidant stressis one of the causes of vascular inflammation, and is also one of themechanisms of progress of arteriosclerosis. (Renard C. et al., DiabetesMetab. 32: 15 (2005)). Vitamin E (γ-tocopherol and α-tocopherol) is oneof the antioxiding agents of a cell membrane, and is transported vialipoprotein into the body. Vitamin E (γ-tocopherol and α-tocopherol) isalso one of the antioxiding agents of the lipoprotein.

Though little is known of the relationship between vitamin E containedin the lipoprotein in the blood and diseases, there are the followingreports in addition to the above:

(3) Yolanda B. et al., Arterioscler. Thromb. Vasc. Biol. 17: 127 (1997);α-tocopherol/cholesterol levels in LDL are enhanced in patients withhyperlipidemia compared to healthy individuals.

Little is also known of the relationship between vitamin E in the bloodand diabetes or coronary artery diseases; however there are thefollowing reports:

(4) Salonen JT et al., BMJ 31: 1124 (1995); α-tocopherol/cholesterollevels are reduced in patients with diabetes compared to healthyindividuals.

(5) Reunanen A. et al., Eur. J. Clin. Nutr. 52(2): 89 (1998); Reducedlevels of α-tocopherol lead to a higher risk of developing diabetes.

(6) Sobczak A. et al., J. Chromatogr. 730, 265 (1999); α-tocopherol andγ-tocopherol levels are enhanced in patients with diabetes compared tohealthy individuals.

(7) Mayer-Davis E J et al., Diabetes Care 25: 2172 (2002); α-tocopherollevels are reduced in patients with diabetes compared to healthyindividuals.

(8) Ohrvall M. et al., J. Intern. Med. 239: 111 (1996); γ-tocopherollevels are reduced in patients with coronary artery diseases compared tohealthy individuals but no significant changes in α-tocopherol. Theα-tocopherol/γ-tocopherol ratio becomes high.

There are no reports on vitamin E in the lipoproteins other than theabove, but since vitamin E are contained in other lipoproteins, researchon the etiology and treatment of arteriosclerosis and diabetes arelikely to make a great progress by comprehensive investigation on theamount of vitamin E in each lipoprotein.

As a conventional method of analyzing vitamin E in the lipoprotein, amethod is known in which after each lipoprotein is separated, vitamin Eare extracted by hexane, the extracted solution is dried, and thenredissolved in methanol etc., and subjected to a reverse phasechromatography (Haidari M. et al., Clin. Chem. 47: 1234 (2001); Feki M.et al., Clin. Chem. 46: 1401 (2000); Yolanda B. et al., ArteriosclerosisThrobosis and Vascular Biology 17: 127 (1997); Teissier E. et al.,Clinical Chemistry 42: 430 (1996)). For the separation of eachlipoprotein, there are known a method comprising ultracentrifugation(Haidari M. et al. (2001), supra; Feki M. et al. (2000), supra; YolandaB. et al. (1997), supra; Teissier E. et al. (1996), supra), a methodcomprising acrylamide electrophoresis, a method comprising gelfiltration chromatography, and a method comprising ion exchangechromatography (Hirowatari Y. et al., J. Lipid Research 44: 1404 (2003);Hirowatari Y. et al., Anal. Biochem. 308: 336 (2002)).

For the separation of each lipoprotein, an ion exchange chromatographyhas been proposed (Hirowatari Y. et al. (2003), supra) that permitssuccessful separation of each lipoprotein utilizing subtle changes inelectric charge by varying the composition of the eluant or theseparation conditions. However, the analysis of vitamin E in eachlipoprotein requires, as described above, complicated steps of, afterseparation of each lipoprotein, extracting vitamin E, concentrating todryness, redissolving, and subjecting to a reverse phase chromatography,and thus it was prone to errors and required a great deal of efforts andtime. Though the measurement of changes in vitamin E in each lipoproteinand in the blood and their use as one of the methods for judging thepathological conditions is important in such diseases as diabetes andcoronary artery diseases in which oxidative stress and the pathologicalconditions are closely related, ultracentrifugation is the onlyavailable method for measuring vitamin E in each lipoprotein, which isan expensive apparatus and the operation is complicated, and thusresearch has not made much progress. Furthermore, for vitamin E in theblood extraction by an organic solvent is necessary, the procedure iscomplicated, and the evaluation result on diabetes is different withresearchers.

DISCLOSURE OF THE INVENTION

Thus the primary object of present invention is to provide a method ofanalyzing vitamin E in lipoproteins that permits the qualitative andquantitative measurement of vitamin Es in a process which is simplifiedto the extent amenable to automatic analysis, and an analyticalapparatus that enables to automatically carry out said analyticalmethod. Furthermore, by using such an analytical method and ananalytical apparatus, the present inventor has found that by usingvitamin E in the lipoprotein as an index, various pathologicalconditions such as the pathological conditions of diabetes, the risk ofa coronary artery disease, and the pathological conditions of myocardialinfarction can be judged.

The present invention made for the above primary objective is asfollows:

(1) A method of analyzing vitamin E components in the lipoprotein, whichmethod comprises subjecting a lipoprotein-containing sample to an ionexchange chromatography to separate the lipoprotein, reacting theseparated lipoprotein to a pretreating solution containing an organicsolvent and a surfactant to liberate vitamin E components, and thensubjecting the liberated vitamin E components to a reverse phasechromatography.

(2) The analytical method according to (1) wherein the pretreatingsolution comprises a 10-50% organic solvent and a 0.2-6.0% surfactant atthe step of reacting with the lipoprotein separated by the ion exchangechromatography.

(3) The analytical method according to (1) or (2) wherein thepretreating solution further comprises 50-150 mmol/L of a caotropic ionat the step of reacting with the lipoprotein separated by the ionexchange chromatography.

(4) An analytical apparatus comprising a sample feeding part forcollecting a given amount of a sample, an ion exchange chromatographypart equipped with an ion exchange column, a reagent mixing part formixing part or all of the eluted solution from the ion exchangechromatography part with the reagent, a reverse phase chromatographypart equipped with a reverse phase column, a detection part for carryingout detection on the eluted solution from the reverse phasechromatography part, and a liquid delivery part for delivering thesample collected at the sample feeding part and the eluant for the ionexchange chromatography, a liquid delivery part for delivering thereagent, and a liquid delivery part for delivering the mixture of theeluted solution from the ion exchange chromatography part and thereagent, and a liquid delivery part for delivering the eluant for thereverse phase chromatography.

After measuring the sera from 20 cases of diabetes and 17 cases ofmyocardial infarction that are highly related to oxidative stress and 20healthy individuals as the control using the above analytical method andanalytical apparatus, we have discovered a method of judging variouspathological conditions such as the pathological conditions of diabeticconditions, the risks of coronary artery diseases, and myocardialinfarction using α-tocopherol/cholesterol as an index.

(5) A method of judging the pathological conditions of diabetes usingthe γ-tocopherol/cholesterol value in the very low density lipoprotein(VLDL).

(6) The method according to (5) wherein when theγ-tocopherol/cholesterol value in the very low density lipoprotein(VLDL) of a subject is lower than the mean value of the healthyindividuals, the subject is judged to have the pathological conditionsof diabetes, and when it is not lower than the mean value of the healthyindividuals, the subject is judged not to have the pathologicalconditions of diabetes.

(7) A method of judging the risk of a coronary artery disease using theγ-tocopherol/cholesterol value in the very low density lipoprotein(VLDL).

(8) The method according to (7) wherein, in cases where the very lowdensity lipoprotein (VLDL) cholesterol value of a subject is lower thanthe mean value of the healthy individuals, when theγ-tocopherol/cholesterol value in the very low density lipoprotein(VLDL) of a subject is lower than the mean value of the healthyindividuals, the subject is judged to have the risk of a coronary arterydisease, and when it is not lower than the mean value of the healthyindividuals, the subject is judged to not be at risk for coronary arterydisease.

(9) A method of judging the pathological conditions of diabetes usingthe α-tocopherol/cholesterol value in the low density lipoprotein (LDL).

(10) The method according to (9) wherein when theα-tocopherol/cholesterol value in the low density lipoprotein (LDL) of asubject is higher than the mean value of the healthy individuals, thesubject is judged to have the pathological conditions of diabetes, andwhen it is not higher than the mean value of the healthy individuals,the subject is judged not to have the pathological conditions ofdiabetes.

(11) A method of judging the pathological conditions of myocardialinfarction using the αtocopherol/cholesterol value in the low densitylipoprotein (LDL).

(12) The method according to (11) wherein when theα-tocopherol/cholesterol value in the low density lipoprotein (LDL) of asubject is higher than the mean value of the healthy individuals, thesubject is judged to have the pathological conditions of myocardialinfarction, and when it is not higher than the mean value of the healthyindividuals, the subject is judged not to have the pathologicalconditions of myocardial infarction.

(13) A method of judging the pathological conditions of diabetes usingthe α-tocopherol/cholesterol value in the very low density lipoprotein(VLDL).

(14) The method according to (13) wherein when theα-tocopherol/cholesterol value in the very low density lipoprotein(VLDL) of a subject is lower than the mean value of the healthyindividuals, the subject is judged to have the pathological conditionsof diabetes, and when it is not lower than the mean value of the healthyindividuals, the subject is judged not to have the pathologicalconditions of diabetes.

(15) A method of judging the risk of a coronary artery disease using theα-tocopherol/cholesterol value in the very low density lipoprotein(VLDL).

(16) The method according to (15) wherein, in cases where the very lowdensity lipoprotein (VLDL) cholesterol value of a subject is lower thanthe mean value of the healthy individuals, when theα-tocopherol/cholesterol value in the very low density lipoprotein(VLDL) of a subject is lower than the mean value of the healthyindividuals, the subject is judged to have the risk of a coronary arterydisease, and when it is not lower than the mean value of the healthyindividuals, the subject is judged not to be at risk for coronary arterydisease.

(17) A method of judging the pathological conditions of diabetes usingthe γ-tocopherol/α-tocopherol ratio in the blood

(18) The method according to (17) wherein when theγ-tocopherol/α-tocopherol ratio in the blood of a subject is lower thanthe mean value of the healthy individuals, the subject is judged to havethe pathological conditions of diabetes, and when it is not lower thanthe mean value of the healthy individuals, the subject is judged not tohave the pathological conditions of diabetes.

Preferably the above methods (5) to (18) may be carried out using anymethod according to the above (1) to (3) or the apparatus according tothe above (4).

As used herein, “lower than the mean value of the healthy individuals”means that the measured α-tocopherol/cholesterol value is lower than themean value of the healthy individuals by 10%, preferably by 20%, morepreferably by 30%, and still more preferably by 50%.

Herein “higher than the mean value of the healthy individuals” meansthat the measured α-tocopherol/cholesterol value is higher than the meanvalue of the healthy individuals by 10%, preferably by 20%, morepreferably by 30%, and still more preferably by 50%.

Since the measurement of vitamin E concentration (γ-tocopherol andα-tocopherol) in each lipoprotein and of total vitamin E concentrationin all the lipoproteins are intended to examine the antioxidant abilityof the lipoprotein, it is necessary to investigate the amount of vitaminE per lipoprotein particle. We have decided to determine the amount ofvitamin E per lipoprotein by dividing by the value of cholesterol whichis an ingredient relatively rich in the lipoprotein. Methods ofcorrection by dividing by cholesterol has generally been used (HaidariM. et al. (2001), supra; Feki M. et al. (2000), supra; Yolanda B. et al.(1997), supra).

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows a first example of the analytical apparatus of the presentinvention.

FIG. 2 shows a second example of the analytical apparatus of the presentinvention.

FIG. 3 shows a third example of the analytical apparatus of the presentinvention.

FIG. 4 is a drawing that shows the constitution of the ion exchangechromatography analytical apparatus of Example 1.

FIG. 5 shows the result of analysis of a serum sample by an ion exchangechromatography.

FIG. 6 shows the result of analysis of a HDL sample by an ion exchangechromatography.

FIG. 7 shows the result of analysis of a LDL sample by an ion exchangechromatography.

FIG. 8 shows the result of analysis of a IDL sample by an ion exchangechromatography.

FIG. 9 shows the result of analysis of a VLDL sample by an ion exchangechromatography.

FIG. 10 shows the result of analysis of a CM sample by an ion exchangechromatography.

FIG. 11 shows the constitution of the reverse phase chromatographyanalytical apparatus of Example 2.

FIG. 12 shows the result of analysis of a vitamin E standard sample by areverse phase chromatography and an electrochemical detection.

FIG. 13 shows the result of analysis of a vitamin E standard sample by areverse phase chromatography and a fluorescent detection.

FIG. 14 shows the result of analysis of a HDL sample by a reverse phasechromatography and an electrochemical detection.

FIG. 15 shows the result of analysis of a HDL sample by a reverse phasechromatography and a fluorescent detection.

FIG. 16 shows the result of analysis of a LDL sample by a reverse phasechromatography and an electrochemical detection.

FIG. 17 shows the result of analysis of a LDL sample by a reverse phasechromatography and a fluorescent detection.

FIG. 18 shows the result of analysis of a IDL sample by a reverse phasechromatography and an electrochemical detection.

FIG. 19 shows the result of analysis of a IDL sample by a reverse phasechromatography and a fluorescent detection.

FIG. 20 shows the result of analysis of a VLDL sample by a reverse phasechromatography and an electrochemical detection.

FIG. 21 shows the result of analysis of a VLDL sample by a reverse phasechromatography and a fluorescent detection.

FIG. 22 shows the result of analysis of a CM sample by a reverse phasechromatography and an electrochemical detection.

FIG. 23 shows the result of analysis of a CM sample by a reverse phasechromatography and a fluorescent detection.

FIG. 24 shows the result of analysis of vitamin E in HDL by anelectrochemical detection.

FIG. 25 shows the result of analysis of vitamin E in HDL by afluorescent detection.

FIG. 26 shows the result of analysis of vitamin E in LDL by anelectrochemical detection.

FIG. 27 shows the result of analysis of vitamin E in LDL by afluorescent detection.

FIG. 28 shows the result of analysis of vitamin E in IDL by anelectrochemical detection.

FIG. 29 shows the result of analysis of vitamin E in IDL by afluorescent detection.

FIG. 30 shows the result of analysis of vitamin E in VLDL by anelectrochemical detection.

FIG. 31 shows the result of analysis of vitamin E in VLDL by afluorescent detection.

FIG. 32 shows the result of analysis of vitamin E in CM by anelectrochemical detection.

FIG. 33 shows the result of analysis of vitamin E in CM by a fluorescentdetection.

FIG. 34 shows the result of analysis of vitamin E in HDL by anelectrochemical detection.

FIG. 35 shows the result of analysis of vitamin E in HDL by afluorescent detection.

FIG. 36 shows the result of analysis of vitamin E in LDL by anelectrochemical detection.

FIG. 37 shows the result of analysis of vitamin E in LDL by afluorescent detection.

FIG. 38 shows the result of analysis of vitamin E in IDL by anelectrochemical detection.

FIG. 39 shows the result of analysis of vitamin E in IDL by afluorescent detection.

FIG. 40 shows the result of analysis of vitamin E in VLDL by anelectrochemical detection.

FIG. 41 shows the result of analysis of vitamin E in VLDL by afluorescent detection.

FIG. 42 shows the result of analysis of vitamin E in CM by anelectrochemical detection.

FIG. 43 shows the result of analysis of vitamin E in CM by a fluorescentdetection.

FIG. 44 shows the γ-tocopherol/cholesterol value in HDL.

FIG. 45 shows the γ-tocopherol/cholesterol value in LDL.

FIG. 46 shows the γ-tocopherol/cholesterol value in IDL.

FIG. 47 shows the γ-tocopherol/cholesterol value in VLDL.

FIG. 48 shows a relationship between the γ-tocopherol/cholesterol valueand VLDL cholesterol in VLDL.

FIG. 49 shows that there is no relationship between VLDL cholesterol andthe γ-tocopherol value in VLDL.

FIG. 50 shows the γ-tocopherol/cholesterol value in CM.

FIG. 51 shows the γ-tocopherol/cholesterol value in the blood.

FIG. 52 shows the α-tocopherol/cholesterol value in HDL.

FIG. 53 shows the α-tocopherol/cholesterol value in LDL.

FIG. 54 shows the α-tocopherol/cholesterol value in IDL.

FIG. 55 shows the α-tocopherol/cholesterol value in VLDL.

FIG. 56 shows a relationship between the α-tocopherol/cholesterol valueand VLDL cholesterol in VLDL.

FIG. 57 shows that there is no relationship between VLDL cholesterol andthe α-tocopherol value in VLDL.

FIG. 58 shows the α-tocopherol/cholesterol value in CM.

FIG. 59 shows the α-tocopherol/cholesterol value in the blood.

FIG. 60 shows the γ-tocopherol value in the blood.

FIG. 61 shows the α-tocopherol value in the blood.

FIG. 62 shows the γ-tocopherol/α-tocopherol value in the blood.

BEST MODE FOR CARRYING OUT THE INVENTION

Lipoproteins are contained in blood (serum). The present invention is ananalytical method to be applied to blood samples, but it is not limitedto blood in the narrow sense as the sample, but may be applied tosamples containing lipoproteins, for example the serum, the plasma, alipoprotein fraction harvested from the blood or serum, or a suspensionobtained by, for example, concentrating said fraction then suspending ina suitable solution, and the like. Vitamin E components to be analyzedin the present invention are mainly α-tocopherol and γ-tocopherol.

The two chromatographic steps of the present invention may preferably becarried out with a column containing a separating agent for use in eachchromatography. According to the present invention, the sample is firstsubjected to an ion exchange chromatography to separate eachlipoprotein. The ion exchange chromatography has a high resolution andcan precisely separate various lipoproteins, and the separating agent(ion exchanger) used may most preferably be an anion exchanger (ananionic ion exchanger) for anions whose resolution is higher than thatof the cation ion exchanger. Various conditions in the ion exchangechromatography such as the amount of the separating agent used (thevolume of an ion exchanger), the composition of the eluant and the flowrate of the eluant may be decided as appropriate depending on the typeand amount of the sample to be analyzed by performing variouspreliminary investigations on the conditions adopted in Examplesdescribed below so that each lipoprotein may be separated and collectedas a different fraction.

To the eluant to be used in an ion exchange chromatography, a caotropicion may preferably be added as shown in Examples, since theprotein-denaturing effect loosens the higher structure of the apoproteinon the surface of lipoprotein particles and thereby can enhance thedestruction of lipoprotein particles and the efficiency of liberatingvitamin E components by use of a surfactant. As the caotropic ion, therecan be illustrated a perchlorate ion, urea, guanidine, a thiocyanateion, an iodine ion and the like. The concentration to be added maydiffer with the strength of caotropic effect of each ion, and for aperchlorate ion or a thiocyanate ion, when mixed with a sample, it is aconcentration that gives 50-150 mmol/L, more preferably 50 mmol/L.

When a high concentration of caotropic ion (300 mmol/L or higher in thecase of a perchlorate ion or a thiocyanate ion) is added to the eluant,the higher structure of the apoprotein may completely be destroyedresulting in the destruction of the structure of the lipoprotein aswell, and thus may cause troubles in the separation of each lipoproteinby an ion exchange chromatography. Thus, when a high concentration ofcaotropic ion is to be used, it is preferred that after separation ofeach lipoprotein by an ion exchange chromatography, a high concentrationof caotropic ion is added and reacted, or a low concentration ofcaotropic ion is added to the eluant in an ion exchange chromatography,and the caotropic ion is additionally added to the above fraction.

In the ion exchange chromatography, after lipoprotein in the sample hasbeen adsorbed to a separating agent, it is subjected to a stepped orgradient elution so as to eluate each lipoprotein based on thedifference in the electric charge in a stepped or gradient elution; inthe case of the stepped elution, the eluted solution may be collected infractions for each step, or in the case of gradient elution, the elutedsolution may be collected in fractions for a given volume. Then, to theentire fractions collected, the pretreating solution may be added at agiven volume ratio, or after selecting the lipoprotein-containingfractions from the collected fractions, the pretreating solution may beadded to the selected fractions at a given volume ratio, and allowed toreact to the lipoprotein. The reaction of the lipoprotein and thepretreating solution may be carried out by merely mixing the two andallowing to stand.

The pretreating solution may comprise an organic solvent for dissolvingvitamin E components and a surfactant for destroying the lipoproteinparticles and liberating vitamin E components therefrom, and, maycomprise, in addition to the above, a reducing agent such as ascorbicacid for preventing the oxidation of vitamin E components, specificallywhen a caotropic ion has not been added to the eluant in an ion exchangechromatography, the pretreating solution may preferably contain acaotropic ion. The caotropic ion may be added in order to denature thehigher structure of apoprotein on the surface of the lipoproteinparticles so as to enhance the efficiency of destroying lipoproteinparticles as well as to prevent the insolubilization of various proteinscontained in the lipoprotein fractions caused by contact with an organicsolvent so as to obviate the need of removing insoluble mattersresulting from protein by centrifugation or filtering with a fine filterprior to subjecting to a reverse phase chromatography described below.Even if a caotropic ion has been added to the eluant in ion exchangechromatography, it may not preclude the addition of a caotropic ion tothe pretreating solution. With regard to the amount mixed of thepretreating solution relative to the lipoprotein-containing fraction,when the lipoprotein fraction eluted from the ion exchange column isexcessively diluted the detection sensitivity in the subsequent reversephase chromatography may be lowered, and depending on the concentrationof a surfactant contained in the pretreating solution, mixing in anexcessive amount may induce foaming, and thus the amount mixed of thepretreating solution relative to the lipoprotein-containing fraction maypreferably be ⅕ to 5 times the amount of the fraction, more preferably ½to twice the amount of the fraction.

As the caotropic ion, there can be illustrated a perchlorate ion, urea,guanidine, a thiocyanate ion, an iodine ion and the like. Theconcentration may vary with the strength of caotropic effect of eachion, and for a perchlorate ion or a thiocyanate ion when mixed with alipoprotein, it is a concentration that gives 50-150 mmol/L, morepreferably 50 mmol/L. The organic solvent may not be specificallylimited as long as it can dissolve vitamin E components, and one or morethan one selected from ethanol, acetonitrile, methanol, isopropanol oracetone may be illustrated. The concentration may vary with the type ofthe organic solvent, and in the case of ethanol, it is a concentrationthat becomes 10 to 50%, more preferably 25% when mixed with thelipoprotein-containing fraction. The surfactant may not be specificallylimited as long as it can destroy the lipoprotein alone or incombination with a caotropic ion, and can liberate vitamin E components,and one or more than one selected from sodium dodecyl sulfate (SDS),poly(oxyethylene)sorbitan monolaulate (Tween 20), Triton X-100, Brij 35or deoxycholic acid. The concentration may vary with the type of thesurfactant, and for SDS, according to the investigation of the presentinventor, it is a concentration that becomes 6.9 to 208 mmol/L (0.2-6.0%in terms of weight), more preferably 100 mmol/L (2.9% in terms ofweight) when mixed with the lipoprotein-containing fraction. The abovedisclosure for the organic solvent, the surfactant in the pretreatingsolution and the concentration of the preferably contained caotropic ionis for the purpose of reference, and depending on the type of theactually selected organic solvent, surfactant and the caotropic ion,modifications may be required. Thus, in the analysis of vitamin Ecomponents based on the present invention, it is preferred that theoptimum range be investigated in advance for the concentration of eachcomponent contained in the pretreating solution.

Vitamin E components liberated from the lipoprotein by the reaction withthe pretreating solution may be subjected to a reverse phasechromatography as a mixed solution of the lipoprotein-containingfraction and the pretreating solution. Vitamin E analysis may be carriedout by a gas chromatography, a thin layer chromatography, NMR etc., butsince a salt-containing eluant is used in separating and eluting eachlipoprotein-containing fraction by an ion exchange chromatography, thepresent invention adopts a reverse phase chromatography that permitsprecise analysis of salt-containing analytes. By subjecting the abovesample mixed with the pretreating solution to a reverse phasechromatography using a eluant containing an organic solvent, vitamin Ecomponents are separated and eluted, and detected by a detector toanalyze each vitamin E. Various conditions such as the amount of theseparating agent, the composition of the eluant, and the flow rate ofthe eluant used are determined as appropriate by performing variouspreliminary investigations including conditions adopted in the Examplesdescribed below depending on the type and amount of samples to besubjected to analysis. After performing an ion exchange chromatographyand before performing a reverse phase chromatography, δ-tocopherol, oneof vitamin E components, may be added as an internal control to enhanceanalytical precision. Though δ-tocopherol is one of vitamin Ecomponents, its amount in the lipoprotein is as small as about onehundredth that of α-tocopherol, and therefore it can be used as aninternal control.

Though, for the detection of vitamin E, a UV-absorption detector, a massdetector, a fluorescence detector, an amperometric electrochemicaldetector, or a coulombic electrochemical detector etc. may be used, buta fluorescence detector, an amperometric detector, or a coulombicelectrochemical detector are preferred because of sufficient detectionsensitivity and ease of maintenance works.

Next, the analytical apparatus of the present invention will beexplained with reference to FIG. 1 to FIG. 3. In the apparatus shown inFIG. 1, the sample feeding part is composed of an autosampler 2 and apipeline; the ion exchange chromatography part is composed of an ionexchange column 1 and a pipeline; the reagent mixing part that mixes theeluted solution from the ion exchange chromatography part and thereagent is composed of a mixer 10 and a branched pipeline 22 connectedimmediately therebefore (upstream side); the reverse phasechromatography part is composed of a reverse phase column 13 and apipeline; the detecting part that performs detection on the elutedsolution from the reverse phase chromatography is composed of afluorescence detector 14, an amperometric detector 15 and a pipeline;the liquid delivery part that delivers the sample and the eluant for theion exchange chromatography is composed of a mixing apparatus 3 for theeluant, liquid delivery pumps 4 and 5 and a pipeline; the liquiddelivery part for delivering the reagent is composed of a liquiddelivery pump 8; and the liquid delivery part for delivering the eluantfor the reverse phase chromatography is composed of pumps 16 and 17. Inthis example, by using a six-way switch valve 11, the delivery of amixture solution of the eluted solution from the ion exchangechromatography part and the reagent is performed, instead of using adedicated liquid delivery pump, by a liquid delivery pump 16 and/or 17for delivering the eluant for the reverse phase chromatography (In theexample shown in FIG. 2, by using two three-way switch valves 20 and 21,the liquid delivery pump 8 for delivering the reagent is also used asthe liquid delivery pump 16 and/or 17 for delivering the eluant for thereverse phase chromatography, and in the example shown in FIG. 3, byusing a six-way switch valve 11, the liquid delivery pump 8 fordelivering the reagent is also used as the liquid delivery pump 16and/or 17 for delivering the eluant for the reverse phasechromatography).

The sample, as it is separately retained in a sample vessel, is carriedto the sample feeding part. On the other hand, the eluant for the ionexchange chromatography is connected, as it is separately retained inthe eluant vessels 6 and 7, via a pipeline to a pump 4 or 5, the reagentto be mixed with the eluted solution from the ion exchangechromatography part is connected, as it is retained in a reagent vessel9, to a pump 8, and the eluant for the reverse phase chromatography isconnected, as it is separately retained in the eluants 18 and 19, via apipeline to a pump 16 or 17. Among the eluted solutions from the ionexchange chromatography part, the eluted solutions which have not beensubjected to the reverse phase chromatography part and for whichdetection at the detection part is complete are discarded as a waste toa suitable waste container.

The sample feeding part is not specifically limited as long as it canautomatically collect a given amount of the sample from the samplevessel etc. carried to a predetermined site of the sample feeding part,and for example a commonly used autosampler such as AS-8020 (trade name,manufactured by TOSOH Corp.) may be used. The sample feeding part mayalso be equipped with an apparatus that automatically carries the samplevessel to the above predetermined site.

An ion exchange column 1 that is mounted to the ion exchangechromatography part is not specifically limited as long as it canseparate each lipoprotein, and for example DEAE-NPR, DEAE-5PW and SP-NPR(all are trade names, manufactured by TOSOH Corp.) may be used. Asdescribed above, specifically an anion exchange column (for exampleDEAE-NPR (trade name, manufactured by TOSOH Corp.)) may preferably beused. On the other hand, a reverse phase column 13 mounted to thereverse phase chromatography part is not specifically limited as long asit can separate vitamin E, and for example ODS-80Ts (trade name,manufactured by TOSOH Corp.)) may be used.

One round of analysis by a reverse phase chromatography takes a givenperiod of time. When one round takes about 40 minutes, as described inThe Examples described below, it is possible that another lipoprotein iseluted from the ion exchange chromatography before the completion of theanalysis of any lipoprotein fraction already eluted from the ionexchange chromatography. Thus, it is preferred that the reverse phasecolumn used may be a column that can curtail the analysis time such as amicrocolumn so that each lipoprotein fraction eluted from the ionexchange chromatography may be sequentially subjected to the reversephase chromatography. It is possible, however, to repeat the sequencethat, as described in The Examples, after the completion of analysis ofa lipoprotein eluted fraction by the reverse phase column, the samesample is fed again to the ion exchange chromatography to collectanother lipoprotein eluted fraction, which is then subjected to thereverse phase column so that vitamin E components in each lipoproteinmay be analyzed.

The reagent mixing part may be constructed so that part or all of theeluted solution from the ion exchange chromatography part may be mixedwith the reagent. In this example, the entire eluted solution from theion exchange chromatography part is mixed with the reagent, and part ofit may be collected by a switch valve 11 and delivered to the reversephase chromatography part. However, in order to minimize the consumptionof the reagent, it is so constructed that the reagent may be mixed withpart of the solution. Thus, the elution time of each lipoprotein fromthe ion exchange column can be predicted if the chromatography conditionis constant, and thus the reagent is delivered by pump 8 in tune withthe predicted timing and in other times the delivery is stopped. Themixing of the reagent may be effected by, for example, merely joiningthe pipeline along which the reagent is delivered with the pipelinealong which the eluted solution from the ion exchange chromatographypart is delivered, but as shown in the example, after joining, mixingmay be performed using a mixing apparatus such as the Static mixer B(trade name, manufactured by TOSOH Corp.), or, for example, afterjoining, the mixture may be passed through a pipeline having a largeinner diameter in which the flow rate is lowered to facilitate themixing of the two, or, for example, pipelines having a rugged surface onthe inner wall or pipelines having varying inner diameters may be used,and the like.

The detection part is not specifically limited as long as it is adetector capable of detecting vitamin E, and for example a UV-absorptiondetector, a mass detector, a fluorescence detector, an amperometricelectrochemical detector, or a coulombic electrochemical detector etc.may be used, but because of sufficient detection sensitivity and ease ofmaintenance works, a fluorescence detector, an amperometricelectrochemical detector, or a coulombic electrochemical detector thatwere used in the above example are preferred.

If the liquid delivery part that delivers the eluant for the ionexchange chromatography uses the eluants 6 and 7 having a different saltconcentration, and the running of the liquid delivery pumps 4 and 5 arecontrolled by a computer etc. to control the amount of the liquiddelivered, eluants having different compositions (salt concentration)can be delivered to the ion exchange column 1 in either of a step orgradient mode. The mixing apparatus 3 is to mix the eluants 6 and 7. Asthe liquid delivery pumps 4 and 5, for example DP-8020 (trade name,manufactured by TOSOH Corp.) may be used, and as the mixing apparatus 3a Static mixer C (trade name, manufactured by TOSOH Corp.) may be used.

For the liquid delivery part for delivering the reagent or the liquiddelivery part for delivering the eluant for the reverse phasechromatography, a pump such as DP-8020 (trade name, manufactured byTOSOH Corp.) can be used. In this example, by using a six-way switchvalve 11, the liquid delivery part for delivering a mixture solution ofthe eluted solution from the ion exchange chromatography part and thereagent and the liquid delivery part for delivering the eluant for thereverse phase chromatography were simplified. However, as shown in FIG.2, there can be illustrated the use of three-way switch valves 20 and21.

In the analysis of vitamin E by the apparatus of FIG. 1, a given amountof the sample collected by an autosampler 2 is delivered together withthe eluant for the ion exchange chromatography with a pump 4 and/or 5 tothe ion exchange column 1 so that the lipoprotein in the sample may beretained on the column. Subsequently, after delivering an amount of thesame eluant, the amount delivered are regulated by pumps 4 and 5 toincrease the salt concentration of the eluant to be subjected to thecolumn, and thus each lipoprotein retained on the column may be eluted.For the fraction of each lipoprotein eluted, the reagent (pretreatingsolution) 9 is delivered by pump 8 to deliver and mix the liquid. Withrespect to the delivering amount of the pretreating solution 9, when thelipoprotein fraction eluted from the ion exchange column is excessivelydiluted the detection sensitivity in the subsequent reverse phasechromatography may be lowered, and depending on the concentration of asurfactant contained in the pretreating solution, delivering in anexcessive amount may induce foaming, and thus the delivering amount ofthe eluant relative to the ion exchange column may preferably be ⅕ to 5times, more preferably ½ to twice the ion exchange column.

Vitamin E components liberated from lipoprotein by mixing with thepretreating solution are then subjected to the reverse phase column 13.By changing the flow path using a six-way switch valve 11, thelipoprotein fraction mixed with the pretreating solution by the liquiddelivery pump 8 is introduced to the sample loop 12, and furthermore bychanging the flow path using the six-way switch valve 11, the mixedsolution is presented to the reverse phase column by the liquid deliverypump 16 and/or 17. The sample loop 12 should be of the volume of themixed solution to be introduced to the reverse phase column. With regardto the presentation of vitamin E components to the reverse phase column13, when two three-way switch valves 20 and 21 are used in combinationas shown in FIG. 2, the three-way switch valve 20 may be operated tochange the flow path to introduce the mixed solution to the reversephase column 13 during the period from the start to the completion ofelution of the lipoprotein fraction from the ion exchange column, andthe three-way switch valve 21 may be operated to change the flow path todiscard the eluant 18 and/or 19. When a six-way switch valve notequipped with a sample loop is used as shown in FIG. 3, the six-wayswitch valve 11 may be switched to introduce the mixed solution to thereverse phase column 13 during the period from the start to thecompletion of elution of the lipoprotein fraction from the ion exchangecolumn. Vitamin E components eluted from the reverse phase column mayeach be detected by a fluorescence detector 14 and an amperometricdetector 15.

As described above, the analytical apparatus in FIGS. 1 to 3 constructedas a analytical apparatus for vitamin E, when an apparatus forautomatically carrying the sample vessel to the predetermined place ofthe sample feeding part and an computer for controlling various partsare added thereto, can automatically analyze vitamin E components in thelipoprotein by merely putting the sample in a vessel and placing it tothe carrying apparatus and the like.

Thus, as a result of utilizing the above analytical method and the aboveanalytical apparatus as shown in the Examples below, it became possibleto judge various pathological conditions such as the pathologicalconditions of diabetic conditions, the risks of coronary arterydiseases, and the pathological conditions of myocardial infarction usingα-tocopherol/cholesterol as an index.

EXAMPLES

The present invention will now be explained in more detail withreference to Examples, but the examples are only embodiments of thepresent invention and do not limit the present invention in any way.

Example 1

A analytical apparatus (FIG. 4) of the following constitution wasconstructed.

Column 1 for the ion exchange chromatography: Two DEAE-NPR columns(trade name, manufactured by TOSOH Corp.), 4.6 mm ID×35 mm, wereconnected in series.

Sample feeding part 2: AS-8020 (trade name, manufactured by TOSOH Corp.)

Mixing apparatus 3: Static mixer C (trade name, manufactured by TOSOHCorp.)

Liquid delivery pumps 4 and 5: DP-8020 (trade name, manufactured byTOSOH Corp.)

Eluant A6: 50 mmol/L Tris+1 mmol/L EDTA 2Na, pH 7.5

Eluant B7: 50 mmol/L Tris+1 mmol/L EDTA 2Na+sodium perchlorate 300mmol/L, pH 7.5

Flow rate of the ion exchange chromatography: 0.5 ml/min

Elution condition of the ion exchange chromatography:

0 minute to 0.05 minute

-   -   Eluant B 10%, eluant A 90%

0.05 minute to 5 minutes

-   -   Eluant B 38%, eluant A 62%

5 minutes to 11 minutes

-   -   Eluant B 44%, eluant A 56%

11 minutes to 16 minutes

-   -   Eluant B 49%, eluant A 51%

16 minutes to 21 minutes

-   -   Eluant B 56%, eluant A 44%

21 minutes to 29 minutes

-   -   Eluant B 100%, eluant A 0%

29 minutes to 40 minutes

-   -   Eluant B 10%, eluant A 90%

Time required for separation: 45 minutes/sample

The separation of the lipoprotein by the above apparatus was tested. Thefractions separated by an ion exchange chromatography from a serum(total cholesterol 159 mg/dL, triglyceride 492 mg/dL) collected afterobtaining the informed consent from an individual who developed ahyperlipidemia and, separately, lipoprotein fractions separated to HDL,LDL, IDL, VLDL, and CM by the ultra centrifugation of the same serumwere mixed with a labelling reagent in which parinaric acid (a reagentthat adsorbs hydrophobic substances and emits fluorescence) was added toa buffer of 90 mmol/L Tris+80 mmol/L boric acid+3 mmol/L EDTA 2Na,pH9.6, to a concentration of 1.4 ng/mL at a flow rate of 0.15 mL/min,and then detected with the FS-8020 (trade name, a fluorescence detectormanufactured by TOSOH Corp., 324 nm, emission wavelength 413 nm).

The results of the serum sample separated by the above apparatus areshown in FIG. 5, and that of preparations by ultracentrifugation of theserum sample are shown in FIGS. 6 to 10. It was confirmed that HDL, LDL,IDL, VLDL, and CM were successfully separated and that albumin and freefatty acids are eluted at the same position as HDL from the comparisonwith the analytical results of pure products (data not shown).

Example 2

A analytical apparatus (FIG. 11) of the following constitution wasconstructed, and the organic solvent, the surfactant and the caotropicion of the pretreating solution were tested.

Column 13 for the reverse phase chromatography: ODS-80Ts (trade name,manufactured by TOSOH Corp.), 4.6 mm ID×150 mm

Sample feeding part 2: AS-8020 (trade name, manufactured by TOSOH Corp.)

Detector 14: FS-8020 (trade name, a fluorescence detector manufacturedby TOSOH Corp.), excitation wavelength 298 nm, emission wavelength 325nm

Liquid delivery pumps 16 and 17: DP-8020 (trade name, manufactured byTOSOH Corp.)

Eluant C18: 30% ethanol+25 mmol/L ammonium nitrate

Eluant D19: 85% ethanol+25 mmol/L ammonium nitrate

Flow rate: 1.0 ml/min

Elution condition:

0 minute to 5 minute

-   -   Eluant C 0%, eluant D 100%

5 minutes to 10 minutes

-   -   Linear gradient from Eluant C 0%, eluant D 100% to Eluant C        100%, eluant D 0%

10 minutes to 33 minutes

-   -   Eluant C 100%, eluant D 0%

33 minutes to 35 minutes

-   -   Linear gradient from Eluant C 100%, eluant D 0% to Eluant C 0%,        eluant D 100%

35 minutes to 45 minutes

-   -   Eluant C 0%, eluant D 100%

Time required for separation: 45 minutes/sample

20 μl of a serum (total cholesterol 215 mg/dL, triglyceride 97 mg/dL)collected after obtaining the informed consent from a healthy individualwas subjected to the apparatus of Example 1, and fractions from 0 minuteto 8 minutes were collected as the HDL fraction and fractions from 8minute to 13 minutes were collected as the LDL fraction. The elutedfractions were tested using SDS, Triton X-100 and Tween 20 as thesurfactant, and ethanol, acetonitrile and methanol as the organicsolvent.

First, the HDL fraction sample was examined (Tables 1 to 3). To 500 μlof the eluted fraction collected, 500 μl of the pretreating solution wasadded, and after mixing at room temperature for 3 seconds, 500 μl of itwas subjected to the above apparatus. The pretreating solution to whichwere added SDS to a post-mixing concentration (final concentration) of12.5-100 mmol/L and furthermore 10 mmol/L of ascorbic acid and 1%phosphoric acid for the purpose of stabilizing lipoproteins was added tothe HDL fraction, and was subjected to the apparatus and analyzed, witha result that, as shown in Table 1, when SDS was 25 mmol/L, bothγ-tocopherol and α-tocopherol gave the highest peak. This reveals thatwith this condition, vitamin E components were efficiently recovered onthe surface of the reverse phase chromatography column.

At the condition of SDS at a final concentration of 25 mmol/L, thepretreating solution to which were added ethanol at a finalconcentration of 12.5-35%, acetonitrile 25%, and methanol 25%, and, forthe purpose of stabilizing lipoproteins, 10 mmol/L of ascorbic acid and1% phosphoric acid was added to the HDL fraction and was subjected tothe apparatus and analyzed, with a result that, as shown in Table 2, thehighest peak was obtained at 25% acetonitrile for γ-tocopherol and at25% methanol for α-tocopherol. This reveals that when SDS is to be used,acetonitrile 25% as the organic solvent is optimum for γ-tocopherol andmethanol 25% is optimum for α-tocopherol.

TABLE 1 Organic solvent Surfactant name name Peak height Peak heightConc. Conc. (mV) (%) No. Name (%) Name (mmol/l) γ-tocopherolα-tocopherol γ-tocopherol α-tocopherol 1 — 0 SDS 0 1.41 3.20 65 62 2 — 0SDS 12.5 1.74 4.25 80 82 3 — 0 SDS 25 2.17 5.17 100 100 4 — 0 SDS 501.84 3.36 85 65 5 — 0 SDS 100 0.97 2.91 45 56

TABLE 2 Organic solvent Surfactant name name Peak height Peak heightConc. Conc. (mV) (%) No. Name (%) Name (mmol/l) γ-tocopherolα-tocopherol γ-tocopherol α-tocopherol 1 — 0 SDS 25 2.17 5.17 89 65 2Ethanol 12.5 SDS 25 1.98 5.20 81 65 3 Ethanol 25 SDS 25 1.62 5.45 66 684 Ethanol 35 SDS 25 1.87 5.00 77 63 5 Acetonitrile 25 SDS 25 2.44 6.57100 82 6 Methanol 25 SDS 25 2.34 8.00 96 100 7 — 0 Triton 0.5% 2.17 3.02— — X-100 8 Ethanol 25 Triton 0.5% 2.04 5.00 — — X-100 9 — 0 Tween 200.5% 1.95 4.09 — — 10 Ethanol 25 Tween 20 0.5% 1.76 4.39 — —

When SDS as the surfactant was replaced with Triton X-100 or Tween 20 ata final concentration of 0.5%, and ethanol was prepared at a finalconcentration of 0% or 25%, as shown in Table 2, the effect of ethanolwas not noted for γ-tocopherol, but for α-tocopherol, the addition ofethanol at both concentrations gave a strong peak height. This revealedthat when SDS is used, acetonitrile or methanol is effective as theorganic solvent for the analysis of α-tocopherol, and when Triton X-100or Tween 20 is used, ethanol is effective.

When the pretreating solution to which were added ethanol at a finalconcentration of 25%, SDS at a final concentration of 0 to 50 mmol/L,and furthermore, for the purpose of stabilizing lipoproteins, and 10mmol/L of ascorbic acid and 1% phosphoric acid were added to the HDLfraction, and subjected to the apparatus and analyzed, with a resultthat, as shown in Table 3, the highest peak was obtained at a conditionof 50 mmol/L of SDS for both of γ-tocopherol and α-tocopherol. Whenethanol 0% and 25% are compared at SDS 50 mmol/L, both γ-tocopherol andα-tocopherol gave a strong peak height in the presence of ethanol,indicating the effect of ethanol addition at 50 mmol/L SDS.

TABLE 3 Organic solvent Surfactant name name Peak height Peak heightConc. Conc. (mV) (%) No. Name (%) Name (mmol/l) γ-tocopherolα-tocopherol γ-tocopherol α-tocopherol 1 Ethanol 25 SDS 0 1.01 2.92 5450 2 Ethanol 25 SDS 12.5 1.27 4.05 68 69 3 Ethanol 25 SDS 25 1.73 5.5193 94 4 Ethanol 25 SDS 50 1.86 5.86 100 100 5 Ethanol 0 SDS 50 1.84 3.3699 57

Then using a serum, the effect of sodium perchlorate contained in thepretreating solution was examined (Table 4). Since the HDL fractioncontained 114 mmol/L of sodium perchlorate derived from the eluant usedfor the ion exchange chromatography, the effect of sodium perchloratewas examined. The above serum from a healthy individual was diluted witheluant A or B used in the ion exchange chromatography in the apparatusof Example 1, and the pretreating solution was added so that at a finalethanol concentration of 25% or 0%, the final concentration of SDS is125 mmol/L, and the final concentrations of ethanol and SDS may be 25%and SDS 25 mmol/L, respectively, and subjected to the apparatus andanalyzed, with a result that, as shown in Table 4, in all conditions astronger peak height was obtained when sodium perchlorate was containedcompared to when there was no sodium perchlorate, and furthermore whenall of ethanol, SDS and sodium perchlorate were contained, the highestpeak height was obtained.

TABLE 4 Organic solvent Surfactant name name Sodium Peak height Conc.Conc. perchlorate (%) No. Name (%) Name (mmol/l) (mmol/l) γ-tocopherolα-tocopherol 1 Ethanol 25 SDS 0 0 4.3 11.4 2 Ethanol 25 SDS 0 150 6.215.5 3 Ethanol 0 SDS 125 0 5.9 8.3 4 Ethanol 0 SDS 125 150 6.3 9.4 5Ethanol 25 SDS 25 0 9.5 25.7 6 Ethanol 25 SDS 25 150 9.9 30.0

Next, the LDL fraction sample was examined (Table 5). The pretreatingsolution in which at a final ethanol concentration of 25%, SDS was madefinal concentrations of 0-50 mmol/L, and at a final SDS concentration of25 mmol/L, the final concentration of acetonitrile or methanol be 25%,and furthermore, for the purpose of stabilizing lipoproteins, 10 mmol/Lof ascorbic acid and 1% phosphoric acid were added was added to the LDLfraction and was subjected to the apparatus and analyzed (the LDL samplecontains 132 mmol/L of sodium perchlorate derived from the eluant usedin the ion exchange chromatography). As a result, similarly to theresult when the HDL fraction was used, as shown in Table 5, the peakheight of both of γ-tocopherol and α-tocopherol became higher as the SDSconcentration became higher, and the highest peak was obtained at thecondition of acetonitrile at a final concentration of 25% and SDS at 25mmol/L.

TABLE 5 Organic solvent Surfactant name name Peak height Peak heightConc. Conc. (mV) (%) No. Name (%) Name (mmol/l) γ-tocopherolα-tocopherol γ-tocopherol α-tocopherol 1 Ethanol 25 SDS 0 0.97 3.69 5762 2 Ethanol 25 SDS 12.5 1.33 4.28 79 71 3 Ethanol 25 SDS 25 1.36 5.3480 89 4 Ethanol 25 SDS 50 1.66 5.45 98 91 5 Acetonitrile 25 SDS 25 1.696.00 100 100 6 Methanol 25 SDS 25 1.55 5.58 92 93

As described above, by mixing the lipoprotein fraction to be subjectedto the reverse phase chromatography with the pretreating solutioncontaining an organic solvent, a surfactant and a caotropic ion, vitaminE components in each fraction can be analyzed in a precise and stablemanner, and it can be seen that even if the pretreating solution shouldbe subjected to the reverse phase chromatography together with thelipoprotein fraction, the analysis of vitamin E components is notaffected.

Example 3

20 μl of a serum (total cholesterol 242 mg/dL, triglyceride 421 mg/dL)collected after obtaining the informed consent from an individual whodeveloped hyperlipidemia was subjected to the apparatus of Example 1,and each lipoprotein fraction eluted was collected as described belowand the separation of the lipoprotein was tested.

HDL fraction: fractions from 0-8 minutes

LDL fraction: fractions from 8-13 minutes

IDL fraction: fractions from 13-19 minutes

VLDL fraction: fractions from 19-24 minutes

Chyromicron (hereinafter referred to as CM) fraction: fractions from24-30 minutes

To 500 μl of each lipoprotein fraction eluted, 500 μl of the pretreatingsolution (ethanol 50%, SDS 250 mmol/L, ascorbic acid 10 mmol/L,phosphoric acid 1%, δ-tocopherol 0.04 μg/mL) was added and mixed, whichwas subjected to the apparatus of Example 2 for the analysis ofγ-tocopherol and α-tocopherol. However, behind the apparatus of Example2, an amperometric electrochemical detector (voltage applied: 600 mV)was connected, and chromatograms were obtained with the two detectors.The results are shown in FIGS. 12 to 23 (odd numbers indicate thechromatograms by fluorescence detection, and even numbers indicate thechromatograms by electrochemical detection). FIGS. 12 and 13 arechromatograms for the standard sample (δ-tocopherol 0.02 μg/mL,γ-tocopherol 0.1 μg/mL, α-tocopherol 0.1 μg/mL) prepared using thepretreating solution diluted two-fold with pure water, from which it canbe seen that δ-tocopherol, γ-tocopherol and α-tocopherol eluted in thisorder giving a favorable analytical result. FIGS. 14 and 15 indicate thechromatograms of the HDL fraction, FIGS. 16 and 17 indicate thechromatograms of the LDL fraction, FIGS. 18 and 19 indicate thechromatograms of the IDL fraction, FIGS. 20 and 21 indicate thechromatograms of the VLDL fraction, and FIGS. 22 and 23 indicate thechromatograms of the CM fraction. In the CM fraction, two peaks ofδ-tocopherol and α-tocopherol, (δ-tocopherol is internal control), weredetected but γ-tocopherol was not, and in the other fractions, all peaksof δ-tocopherol, δ-tocopherol and α-tocopherol were detected. From thepeak height of the fluorescence detector, the concentrations ofγ-tocopherol and α-tocopherol in each lipoprotein in the serum werecalculated and shown in Table 6. In calculation, 6-tocopherol added tothe pretreating solution was used as the internal control to calculatethe recovery in each analysis to correct for the results of analysis,and dilution due to fraction separation and the pretreating solutionhave been taken into consideration. The concentrations of γ-tocopheroland α-tocopherol in the serum are the sum of concentrations in eachlipoprotein fraction.

TABLE 6 γ-tocopherol (μg/mL) α-tocopherol (μg/mL) HDL 0.21 1.57 LDL 0.393.96 IDL 0.25 3.00 VLDL 0.33 4.66 Chylomicrons 0.00 0.57 Serum 1.1813.75

Example 4

20 μl of a serum (total cholesterol 203 mg/dL, triglyceride 215 mg/dL)collected after obtaining the informed consent from an individual whodeveloped hyperlipidemia was subjected to the apparatus below (theapparatus construction is the same as that shown in FIG. 4, and theeluant composition and the elution condition were modified), and at aninterval of 2 minutes from the introduction of the sample to 34 minutes,17 samples were collected, and γ-tocopherol and α-tocopherol in eachlipoprotein were analyzed by the apparatus and the method in Example 3.The cholesterol concentration for each fraction was determined using anenzyme solution containing cholesterol esterase and cholesterol oxidase(Cholesterol E test, trade name, manufactured by Wako Pure ChemicalIndustries, Ltd.).

Column 1 for the ion exchange chromatography: Two DEAE-NPRs (trade name,manufactured by TOSOH Corp.), 4.6 mm ID×35 mm, were connected in series.

Sample feeding part 2: AS-8020 (trade name, manufactured by TOSOH Corp.)

Mixing apparatus 3: Static mixer C (trade name, manufactured by TOSOHCorp.)

Liquid delivery pumps 4 and 5: DP-8020 (trade name, manufactured byTOSOH Corp.)

Eluant A: 50 mmol/L Tris+1 mmol/L EDTA 2Na+100 mmol/L sodiumthiocyanate, pH 7.5

Eluant B: 50 mmol/L Tris+1 mmol/L EDTA 2Na+100 mmol/L sodiumthiocyanate+500 mmol/L sodium nitrate, pH 7.5

Flow rate of the ion exchange chromatography: 0.5 ml/min

Elution condition of the ion exchange chromatography:

0 minute to 24 minutes

-   -   Linear gradient from Eluant B 10%, eluant A 90% to Eluant B 50%,        eluant A 50%

20 minutes to 25 minutes

-   -   Eluant B 50%, eluant A 50%

25 minutes to 30 minutes

-   -   Eluant B 100%, eluant A 0%

30 minutes to 40 minutes

-   -   Eluant B 10%, eluant A 90%

The concentrations of γ-tocopherol, α-tocopherol and cholesterol in eacheluted fraction are shown in Table 7. Fractions 6 and 7 correspond toHDL, fractions 8 and 9 correspond to LDL, fractions 10 and 11 correspondto IDL, fractions 12 and 13 correspond to VLDL, and fractions 13 and 14correspond to CM. Thus, even when lipoprotein was eluted in a linergradient manner in the ion exchange chromatography, the favorableanalysis of vitamin E components in the lipoprotein was attained.

TABLE 7 Tocopherol (ng/ml) Fraction No. γ α Cholesterol (mg/dl) 1 0.000.00 1.6 2 0.00 0.42 0.8 3 0.00 0.00 0.8 4 0.00 0.10 0.0 5 0.00 0.53 0.86 0.00 2.45 4.0 7 0.59 8.54 16.0 8 2.84 29.90 71.2 9 1.83 21.69 44.8 100.71 10.68 16.0 11 0.69 13.33 16.0 12 1.37 20.58 21.6 13 0.79 14.14 12.814 0.00 7.41 5.6 15 0.00 8.30 7.2 16 0.00 1.48 0.0 17 0.00 0.77 0.0

Example 5

As the analytical apparatus of the present invention shown in FIG. 1,specifically the following apparatus was constructed. 20 μl of a serum(total cholesterol 203 mg/dL, triglyceride 215 mg/dL) collected afterobtaining the informed consent from an individual who developedhyperlipidemia was subjected to this apparatus to analyze vitamin Es. Inthis example, instead of determining vitamin E components in eachlipoprotein by the one time introduction of the sample, but byintroducing the sample for a total of five times, vitamin E componentsin each lipoprotein was analyzed.

Column 1 for the ion exchange chromatography: Two DEAE-NPR columns(trade name, manufactured by TOSOH Corp.), 4.6 mm ID×35 mm, wereconnected in series.

Sample feeding part 2: AS-8020 (trade name, manufactured by TOSOH Corp.)

Mixing apparatus 3: Static mixer C (trade name, manufactured by TOSOHCorp.)

Liquid delivery pumps 4 and 5: DP-8020 (trade name, manufactured byTOSOH Corp.)

Eluant A6: 50 mmol/L Tris+1 mmol/L EDTA 2Na, pH 7.5

Eluant B7: 50 mmol/L Tris+1 mmol/L EDTA 2Na+sodium perchlorate 300mmol/L, pH 7.5

Flow rate of the ion exchange chromatography: 0.5 ml/min

Elution condition of the ion exchange chromatography:

0 minute to 0.05 minute

-   -   Eluant B 10%, eluant A 90%

0.05 minute to 5 minutes

-   -   Eluant B 38%, eluant A 62%

5 minutes to 11 minutes

-   -   Eluant B 44%, eluant A 56%

11 minutes to 16 minutes

-   -   Eluant B 49%, eluant A 51%

16 minutes to 21 minutes

-   -   Eluant B 56%, eluant A 44%

21 minutes to 29 minutes

-   -   Eluant B 100%, eluant A 0%

29 minutes to 40 minutes

-   -   Eluant B 10%, eluant A 90%

Liquid delivery pump 8: DP-8020 (trade name, manufactured by TOSOHCorp.)

Pretreating solution 9: 100 mmol/L SDS+50% ethanol

Mixing apparatus 10: Static mixer B (trade name, manufactured by TOSOHCorp.)

Flow rate of the pretreating solution: 0.25 ml/min

Six-way switch valve 11: Six-way electromagnetic valve

-   -   Timing of switching (switching of the flow path from OFF to ON)    -   For HDL elution fraction, for 2 minutes from 7 minutes    -   For LDL elution fraction, for 2 minutes from 11 minutes    -   For IDL elution fraction, for 2 minutes from 17 minutes    -   For VLDL elution fraction, for 2 minutes from 22 minutes    -   For CM elution fraction, for 2 minutes from 27 minutes

Volume of the sample loop 12: 1.5 ml volume

Column 13 for the reverse phase chromatography: ODS-80Ts (trade name,manufactured by TOSOH Corp.), 4.6 mm ID×150 mm

Detector 14: FS-8020 (trade name, a fluorescence detector manufacturedby TOSOH Corp., excitation wavelength 298 nm, emission wavelength 325nm).

Detector 15: EC-8020 (trade name, a amperometric electrochemicaldetector manufactured by TOSOH Corp., applied voltage 600 mV).

Liquid delivery pumps 16 and 17: DP-8020 (trade name, manufactured byTOSOH Corp.)

Eluant C18: 30% ethanol+25 mmol/L ammonium nitrate

Eluant C19: 85% ethanol+25 mmol/L ammonium nitrate

Flow rate of the reverse phase chromatography: 1.0 ml/min

Elution condition of the reverse phase chromatography:

-   -   Initially, eluant C 100%, D 0%    -   Five minutes after the switch valve 11 turned ON, change to        eluant C 100%, D 0%    -   34 minutes after the switch valve 11 turned ON, change to eluant        C 0%, D 100%

Time required for analysis: 46 minutes/sample

The results of analysis of the HDL elution fraction are shown in FIGS.24 and 25, those of the LDL elution fraction are shown in FIGS. 26 and27, those of the IDL elution fraction are shown in FIGS. 28 and 29,those of the VLDL elution fraction are shown in FIGS. 30 and 31, andthose of the CM elution fraction are shown in FIGS. 32 and 33. In eachfigure, even numbers indicate the chromatograms by electrochemicaldetection and odd numbers indicate the chromatograms by fluorescencedetection. As can be seen from each figure, γ-tocopherol andα-tocopherol were successfully detected for each lipoprotein fraction.

Example 6

Except that the composition of the pretreating solution was 50 mmol/Lsodium perchlorate+1% Triton X100+75% ethanol, the same apparatus andthe method as that of Example 5 were used to carry out the analysis. Theresults of analysis of the HDL elution fraction are shown in FIGS. 34and 35, those of the LDL elution fraction are shown in FIGS. 36 and 37,those of the IDL elution fraction are shown in FIGS. 38 and 39, those ofthe VLDL elution fraction are shown in FIGS. 40 and 41, and those of theCM elution fraction are shown in FIGS. 42 and 43. In each figure, evennumbers indicate the chromatograms by electrochemical detection and oddnumbers indicate the chromatograms by fluorescence detection. As can beseen from each figure, γ-tocopherol and α-tocopherol were successfullydetected for each lipoprotein fraction.

As a risk factor for arteriosclerosis, oxidized lipoprotein isattracting much attention. A major antioxidant contained in thelipoprotein is vitamin E (mainly α-tocopherol and γ-tocopherol), and theanalytical method of the present invention for vitamin E in thelipoprotein can analyze the amount of vitamin E in each lipoprotein inthe blood, and thus will be able to obtain important finding inunderstanding the mechanism of arteriosclerosis. If the relationshipbetween arteriosclerosis and vitamin E in the lipoprotein wereelucidated, the analytical method of the present invention would bepreferred for the diagnosis of arteriosclerosis and monitoring oftherapeutic effect.

The analytical apparatus of the present invention can analyze vitamin Eetc. automatically and thus in a short period of time, and besides beingan automatic analysis, there is very little chance of generating errors.

Compared to the constitution in the prior art that after separating eachlipoprotein, vitamin E components are extracted, concentrated todryness, redissolved, and subjected to a reverse phase chromatography,the constitution that after separating each lipoprotein using an ionexchange chromatography, the separated lipoprotein is reacted with apretreating solution containing an organic solvent and a surfactantthereby to liberate vitamin E components, and the liberated vitamin Ecomponents are subjected to a reverse phase chromatography is a simpleconstitution per se and is characterized by a little chance ofgenerating errors. Specifically, the prior art comprised complicated anderror-prone manual works that for each lipoprotein fraction vitamin Ecomponents were manually extracted, concentrated to dryness, andredissolved, but in the present invention, these steps have beenreplaced with a simple step of adding and mixing and reacting thepreviously prepared pretreating solution.

The prior art comprised the complicated steps, which require manualworks of extraction of vitamin E components, concentration to drynessand redissolution, and thus the automatization of the procedure wasdifficult. In the analysis of the present invention, however, theseparating agent used in the ion exchange chromatography and the reversephase chromatography can be loaded in columns for performing theanalysis, and thus the step of mixing the pretreating solution to theeluted solution from the ion exchange chromatography replacing the stepsof extraction of vitamin E components, concentration to dryness andredissolution can be automated in a process in which the eluted solutionof the ion exchange chromatography is subjected to the reverse phasechromatography. As a result, the analytical method of the presentinvention can be easily automated by an analytical apparatus comprisinga sample feeding part for collecting a given amount of a sample, an ionexchange chromatography part equipped with an ion exchange column, areagent mixing part for mixing part or all of the eluted solution fromthe ion exchange chromatography part with the reagent, a reverse phasechromatography part equipped with a reverse phase column, a detectionpart for carrying out detection on the eluted solution from the reversephase chromatography part, and a liquid delivery part for delivering thesample collected at the sample feeding part and the eluant for the ionexchange chromatography, a liquid delivery part for delivering thereagent, and a liquid delivery part for delivering the mixture of theeluted solution from the ion exchange chromatography part and thereagent, and a liquid delivery part for delivering the eluant for thereverse phase chromatography, and thus the analysis time and efforts canbe curtailed.

Example 7

α-tocopherol and γ-tocopherol in the HDL, LDL, IDL, VLDL, chylomicron inthe sera of 17 healthy individuals (Healthy), 20 patients with diabetes(Diabetes) and 17 patients with myocardial infarction (AMI) weredetermined by the method described in Example 3. Also, using the ionexchange chromatography method (Hirowatari Y. et al., J. Lipid Res. 44:1404 (2003)), cholesterol in each lipoprotein was separately determined,and α-tocopherol and γ-tocopherol per cholesterol were calculated.

By summing up α-tocopherol or γ-tocopherol in all lipoproteins, thetotal vitamin E concentration (α-tocopherol or γ-tocopherolconcentrations) in all lipoproteins in the blood was calculated. Andseparately, cholesterol in each lipoprotein was determined, and summedup to calculate cholesterol in the blood, and α-tocopherol andγ-tocopherol per cholesterol were calculated. The results are shown infigures and tables. In the figures, the bar and the square indicate thecenter value (50 percentile) and 25-75 percentile (IQR) in anonparametric statistics.

The γ-tocopherol/cholesterol values (γ-tocopherol value per cholesterol)in the HDL are shown in FIG. 44 and Table 8.

TABLE 8 γ-tocopherol/ cholesterol HDL LDL IDL VLDL CM Serum mmol/molmmol/mol mmol/mol mmol/mol mmol/mol mmol/mol Diabetes Mean 1.23 0.403.17 2.39 11.04 0.90 SD 0.49 0.20 1.64 2.43 5.31 0.28 AMI Mean 1.00 0.374.82 6.91 15.71 0.97 SD 0.53 0.15 2.49 6.16 9.39 0.31 Healthy Mean 1.200.34 6.25 5.05 17.42 1.03 SD 0.72 0.15 3.96 3.76 11.49 0.30 SignificantHealthy vs 0.897 0.269 0.007 0.018 0.047 0.199 test, DiabetesNonparametric Healthy vs 0.364 0.532 0.218 0.299 0.637 0.584 AMI

Diabetes (mean value: 1.23 mmol/mol), myocardial infarction (mean value:1.00 mmol/mol) and the healthy individuals (mean value: 1.20 mmol/mol)exhibited similar values, but some patients with myocardial infarctionexhibited values as low as 0.5 mmol/mol or less, which is obviously lowcompared to the healthy individuals. The presence of myocardialinfarction patients with low values compared to the healthy individualsis thought to indicate that γ-tocopherol in the HDL was decomposed byoxidative stress.

The γ-tocopherol/cholesterol values in the LDL are shown in FIG. 45 andTable 8. Diabetes (mean value: 0.40 mmol/mol), myocardial infarction(mean value: 0.37 mmol/mol) and the healthy individuals (mean value:0.34 mmol/mol) exhibited similar values.

The γ-tocopherol/cholesterol values in the IDL are shown in FIG. 46 andTable 8. Diabetes (mean value: 3.17 mmol/mol) was significantly(p=0.007) lower than the healthy individuals (mean value: 6.25mmol/mol), indicating that γ-tocopherol in the IDL was decomposed byoxidative stress, a pathological condition carried by diabetes. Inmyocardial infarction as well, though there were no significantdifference (p=0.218), the mean value of 4.82 mmol/mol was lower than thehealthy individuals, which is also due to oxidative stress. Criteria forjudgment of significant difference is that: between the values of thehealthy individuals and those of patients, a Welch's t test (t testintended for two samples with unequal variance) is performed, and whenthe p-value is less than 0.05 it was judged to be significantlydifferent, and otherwise judged to be no significant difference (thesame hereinafter).

The γ-tocopherol/cholesterol values in the VLDL are shown in FIG. 47 andTable 8. Diabetes (mean value: 2.39 mmol/mol) was significantly(p=0.018) lower than the healthy individuals (mean value: 5.05mmol/mol), indicating that γ-tocopherol in the VLDL was decomposed byoxidative stress, a pathological condition carried by diabetes. Thisdemonstrates that by examining the γ-tocopherol/cholesterol value in theVLDL, a pathological condition related to oxidative stress of diabetescan be judged.

Also, the relationship with VLDL cholesterol is shown in FIG. 48. Inpatients with myocardial infarction which is a coronary artery disease,there is no overall decline compared to the healthy individuals, but thedistribution was wider, the 25 percentile value (1.31 mmol/mol) of thepatients with myocardial infarction was lower than that (1.91 mmol/mol)of the healthy individuals. VLDL cholesterol (Hubert H B et al., Am. J.Epidemiol. 125: 812 (1987)) has been demonstrated to be a risk factorfor coronary artery diseases, and the relationship between this VLDLcholesterol and the γ-tocopherol/cholesterol value in the VLDL wasinvestigated. It can be seen that as VLDL cholesterol tends to becomehigh, not only in the patients with myocardial infarction but in thepatients with diabetes and in the healthy individuals, theγ-tocopherol/cholesterol value in the VLDL sharply drops. There was norelationship between VLDL cholesterol and the γ-tocopherol/cholesterolvalue in the VLDL (FIG. 49). Among the healthy individuals, those havinga γ-tocopherol/cholesterol value in the VLDL of 3 mmol/mol or less hadthe VLDL cholesterol value of 9 mg/dL or higher, or individuals having ahigh risk of myocardial infarction. Considering this result, it may beassumed that there are individuals having a high risk of myocardialinfarction among the healthy individuals. Based on this, it can be saidthat the γ-tocopherol/cholesterol value in the VLDL can precisely judgethe risk of a coronary artery disease when VLDL cholesterol isrelatively low. Taken together that the value in diabetes which is apathological condition known to become a high risk of arterioscleroticdiseases such as coronary artery disease was low, it can be said thatthe γ-tocopherol/cholesterol value in the VLDL can be used to judge therisk of a coronary artery disease.

The γ-tocopherol/cholesterol values in the CM are shown in FIG. 50 andTable 8. Diabetes (mean value: 11.04 mmol/mol) was significantly(p=0.047) lower than the healthy individuals (mean value: 17.42mmol/mol), indicating that γ-tocopherol in the CM was decomposed byoxidative stress, a pathological condition carried by diabetes. Thoughthere was no significant difference (p=0.637) seen for myocardialinfarction either, the mean value (mean value: 5.71 mmol/mol) was lowerthan the healthy individuals, which is also thought to be due todecomposition by oxidative stress.

The γ-tocopherol/cholesterol values in the blood are shown in FIG. 51and Table 8. Diabetes (mean value: 0.90 mmol/mol) and myocardialinfarction (mean value: 0.97 mmol/mol) were not significantly different(p=0.199 and p=0.584), but were lower than the healthy individuals (meanvalue: 1.03 mmol/mol). In FIG. 51, patients with diabetes and patientswith myocardial infarction having values evidently lower than thehealthy individuals can be recognized though the number of the patientsis small. This indicates that the γ-tocopherol in the blood wasdecomposed by oxidative stress, a pathological condition carried bydiabetes and myocardial infarction.

The α-tocopherol/cholesterol values (α-tocopherol value per cholesterol)in the HDL are shown in FIG. 52 and Table 9.

TABLE 9 α tocopherol/cholesterol HDL LDL IDL VLDL CM Serum mmol/molmmol/mol mmol/mol mmol/mol mmol/mol mmol/mol Diabetes Mean 7.58 3.3315.33 13.46 45.37 5.51 SD 3.26 2.20 9.89 15.05 28.19 1.98 AMI Mean 9.303.94 24.39 26.47 55.39 6.83 SD 7.36 1.58 14.84 21.26 35.18 2.54 HealthyMean 6.09 2.01 20.14 17.51 64.73 4.71 SD 1.31 0.45 7.70 12.20 53.16 1.18Significant Healthy vs 0.072 0.016 0.106 0.373 0.190 0.136 test,Diabetes Nonparametric Healthy vs 0.095 0.0001 0.305 0.144 0.551 0.005AMI

When diabetes (mean value: 7.58 mmol/mol) and myocardial infarction(mean value: 9.30 mmol/mol) were compared to healthy individuals (meanvalue: 6.09 mmol/mol), there were no significant differences (p=0.072and p=0.095), but showed a tendency toward higher values. Since HDL isan important lipoprotein responsible for the central role in lipidmetabolism, this is thought that the content of α-tocopherol in the HDLincreased as a protection against damages due to oxidative stress, apathological condition carried by diabetes and myocardial infarction.

The α-tocopherol/cholesterol values in the LDL are shown in FIG. 53 andTable 9. Diabetes (mean value: 3.33 mmol/mol) was significantly(p=0.016) higher than the healthy individuals (mean value: 2.01mmol/mol). Since LDL is an important lipoprotein responsible for thecentral role in lipid metabolism, this is thought that the content ofα-tocopherol in the LDL increased as a protection against damages due tooxidative stress, a pathological condition carried by diabetes. Thus, itcan be said that the α-tocopherol/cholesterol value in the LDL can beused to judge the pathological conditions related to oxidative stress ofdiabetes.

Myocardial infarction (mean value: 3.94 mmol/mol) was also significantly(p=0.0001) higher than the healthy individuals (mean value: 2.01mmol/mol). Since LDL is an important lipoprotein responsible for thecentral role in lipid metabolism, this is thought that the content ofα-tocopherol in the LDL increased as a protection against damages due tooxidative stress, a pathological condition carried by myocardialinfarction. Thus, it can be said that the α-tocopherol/cholesterol valuein the LDL can be used to judge the pathological conditions related tooxidative stress of myocardial infarction.

The α-tocopherol/cholesterol values in the IDL are shown in FIG. 54 andTable 9. It can be seen that though the significant difference (p=0.106)was small, diabetes (mean value: 15.33 mmol/mol) was obviously lowerthan the healthy individuals (mean value: 20.14 mmol/mol). Thisindicates that α-tocopherol in the IDL was decomposed by oxidativestress, a pathological condition carried by diabetes. Myocardialinfarction (mean value: 24.39 mmol/mol) was higher than the healthyindividuals (mean value: 20.14 mmol/mol), but in the values of patientswith myocardial infarction, variation between higher patients and lowerpatients was great compared to the healthy individuals. In fact, the 25percentile value of 13.7 mmol/mol of the patients with myocardialinfarction was lower than the 25 percentile value of 15.7 mmol/mol ofthe healthy individuals. Considering the possibility that there may besome healthy individuals in whom α-tocopherol in the IDL has beendecomposed by oxidative stress, the decomposition of α-tocopherol in theIDL due to oxidative stress should be more advanced in patients withmyocardial infarction having a value lower than the percentile value of15.7 mmol/mol of the healthy individuals.

The α-tocopherol/cholesterol values in the VLDL are shown in FIG. 55 andTable 9. Though there is no significant difference (p=0.373), diabetes(mean value: 13.46 mmol/mol) was obviously lower than the healthyindividuals (mean value: 17.51 mmol/mol), indicating that α-tocopherolin the VLDL was decomposed by oxidative stress, a pathological conditioncarried by diabetes. Thus, it can be said that theα-tocopherol/cholesterol value in the VLDL can judge the pathologicalcondition related to oxidative stress of diabetes.

Also, the relationship with VLDL cholesterol is shown in FIG. 56. Inpatients with myocardial infarction which is a coronary artery disease,there is no overall decline compared to the healthy individuals. Butthis result is possibly because some healthy individuals are expected tohave a high risk of myocardial infarction. Then, VLDL cholesterol hasbeen demonstrated to be a risk factor for coronary artery diseases(Hubert H B et al., (1987), supra), and the relationship between thisVLDL cholesterol and the α-tocopherol/cholesterol value in the VLDL wasinvestigated. It can be seen that as VLDL cholesterol tends to becomehigh, not only in the patients with myocardial infarction but inpatients with diabetes and the healthy individuals, theα-tocopherol/cholesterol value in the VLDL sharply drops. There was norelationship between the VLDL cholesterol and theα-tocopherol/cholesterol value in the VLDL (FIG. 57). Among the healthyindividuals, those having a α-tocopherol/cholesterol value of 7 mmol/molor less in the VLDL had high VLDL cholesterol values of 15 mg/dL orhigher, indicating that they are individuals having a high risk ofmyocardial infarction. Based on this, it can be said that theα-tocopherol/cholesterol value in the VLDL can precisely judge the riskof a coronary artery disease when VLDL cholesterol is low. Takentogether that the value in diabetes which is a pathological conditionknown to become a high risk of arteriosclerotic diseases such ascoronary artery disease was low, it can be said that theα-tocopherol/cholesterol value in the VLDL can be used to judge the riskof a coronary artery disease.

The α-tocopherol/cholesterol values in the CM are shown in FIG. 58 andTable 9. Though there is no significant difference (P=0.190), diabetes(mean value: 45.37 mmol/mol) tended to be lower than the healthyindividuals (mean value: 64.73 mmol/mol), indicating that α-tocopherolin the CM was decomposed by oxidative stress, a pathological conditioncarried by diabetes. For myocardial infarction, the significantdifference p=0.551 was further lower than for diabetes, and the meanvalue of 55.39 mmol/mol was lower than the healthy individuals, which isalso thought to be due to decomposition by oxidative stress.

The α-tocopherol/cholesterol values in the blood are shown in FIG. 59and Table 9. With the significant difference being p=0.005, myocardialinfarction (mean value: 6.83 mmol/mol) was evidently higher than thehealthy individuals (mean value: 4.71 mmol/mol), and this is thoughtthat LDL and HDL which are major lipoproteins in the blood enhanced thecontent of α-tocopherol as a protection against damages due to oxidativestress, a pathological condition carried by diabetes. For diabetes,though there was no significant difference (P=0.136), the value washigher than the healthy individuals (mean value: 4.71 mmol/mol), andthis is also thought that LDL and HDL which are major lipoproteins inthe blood enhanced the content of α-tocopherol as a protection againstdamages due to oxidative stress, a pathological condition carried bydiabetes.

For the healthy individuals and diabetes, the α-tocopherol value and theγ-tocopherol value in the blood were compared (FIGS. 60, 61). Theα-tocopherol value (mean value: 13.63 μg/ml) of the patients withdiabetes was evidently higher than the healthy individuals (mean value:10.70 μg/ml), and the mean value of γ-tocopherol was slightly lower inpatients with diabetes (mean value: 2.15 μg/ml) compared to the healthyindividuals (mean value 2.23 μg/ml), but in the overall distribution,some patients with diabetes had a high value compared to the healthyindividuals, and the central value is slightly higher (healthyindividuals: central value 2.07 μg/ml, patients with diabetes: centralvalue 2.26 μg/ml). This phenomenon of high values of vitamin E(α-tocopherol and γ-tocopherol) is thought to be one of the protectivemechanism against oxidative stress occurring in the body of patientswith diabetes.

Also, there is a phenomenon that as compared to α-tocopherol,γ-tocopherol is more vulnerable to decomposition by radicals generatedby oxidative stress. Also, it is known that α-tocopherol andγ-tocopherol are similarly absorbed by the intestine, transported viachylomicron to the liver or part thereof is to the peripheral cells, andin the liver by the vitamin E-transporting protein, a certain amount ofα-tocopherol and γ-tocopherol are contained in the VLDL particles andliberated into the blood so as to be transported to the peripheral cellsin the body. From a similar mechanism, the ratio of α-tocopherol andγ-tocopherol absorbed in the intestine and liberated from the liver inthe blood is estimated to be almost constant. Based on these phenomena,it was predicted an investigated that the ratio of γ-tocopherol toα-tocopherol would be an excellent index that reflects the pathologicalconditions related to oxidative stress of diabetes. The results areshown in FIG. 62 and Table 10. The ratio of γ-tocopherol/α-tocopherol inthe blood is, as predicted, significantly (p=0.013) lower in patients(mean value: 0.17) with diabetes than the healthy individuals (meanvalue: 0.22), indicating that it can be used to judge the pathologicalconditions related to oxidative stress of diabetes.

TABLE 10 Serum γ tocopherol α tocopherol γ tocopherol/ (μg/ml) (μg/ml) αtocopherol Diabetes Mean 2.15 13.63 0.17 SD 0.51 3.74 0.05 AMI Mean 1.8213.59 0.15 SD 0.42 5.54 0.06 Healthy Mean 2.23 10.70 0.22 SD 0.58 2.410.06 Significant Healthy vs 0.661 0.007 0.013 test, Non- Diabetesparametric Healthy vs 0.023 0.061 0.005 AMI

Also, the result that in coronary artery diseases, α-tocopherol isslightly higher than the healthy individuals although not significant(p=0.061), was agreed that γ-tocopherol/α-tocopherol ratio issignificantly (P=0.005) low is largely consistent with the contentsdescribed in Ohrvall M. et al. (1996), supra.

1. A method of judging as to whether a subject is suffering fromdiabetes using a γ-tocopherol/cholesterol value in very low densitylipoprotein (VLDL), comprising the steps of: obtaining a samplecontaining VLDL from said subject, measuring the γ-tocopherol andcholesterol values in the VLDL contained in said sample, and thencalculating the γ-tocopherol/cholesterol value in the VLDL by dividingsaid measured γ-tocopherol value by said measured cholesterol value,wherein when the γ-tocopherol/cholesterol value in the VLDL of thesubject is lower than a mean γ-tocopherol/cholesterol value in healthyindividuals who are not suffering from diabetes, the subject is judgedto suffer from diabetes, and when it is not lower than the meanγ-tocopherol/cholesterol value of the healthy individuals, the subjectis judged to be not suffering from diabetes.
 2. A method of judging asto whether a subject has the risk of a coronary artery disease using aγ-tocopherol/cholesterol value in very low density lipoprotein (VLDL),comprising the steps of: obtaining a sample containing VLDL from saidsubject, measuring the γ-tocopherol and cholesterol values in the VLDLcontained in said sample, and then calculating theγ-tocopherol/cholesterol value in the VLDL by dividing said measuredγ-tocopherol value by said measured cholesterol value, wherein, when theγ-tocopherol/cholesterol value in the VLDL of the subject is lower thana mean γ-tocopherol/cholesterol value in healthy individuals who are notsuffering from a coronary artery disease, the subject is judged to havethe risk of a coronary artery disease, and when it is not lower than themean γ-tocopherol/cholesterol value of the healthy individuals, thesubject is judged not to have the risk of a coronary artery disease. 3.A method of judging as to whether a subject is suffering from myocardialinfarction using a α-tocopherol/cholesterol value in low densitylipoprotein (LDL), comprising the steps of: obtaining a samplecontaining LDL from said subject, measuring the α-tocopherol andcholesterol values in the LDL contained in said sample, and thencalculating the α-tocopherol/cholesterol value in the LDL by dividingsaid measured α-tocopherol value by said measured cholesterol value,wherein when the α-tocopherol/cholesterol value in the LDL of thesubject is higher than a mean α-tocopherol/cholesterol value in healthyindividuals who are not suffering from myocardial infarction, thesubject is judged to suffer from myocardial infarction, and when it isnot higher than the mean α-tocopherol/cholesterol value of the healthyindividuals the subject is judged to be not suffering from myocardialinfarction.
 4. A method of judging as to whether a subject has the riskof a coronary artery disease using a α-tocopherol/cholesterol value invery low density lipoprotein (VLDL) comprising the steps of: obtaining asample containing VLDL from said subject, measuring the α-tocopherol andcholesterol values in the VLDL contained in said sample, and thencalculating the α-tocopherol/cholesterol value in the VLDL by dividingsaid measured α-tocopherol value by said measured cholesterol value,wherein, in cases where either the measured cholesterol value in theVLDL of the subject is lower than a mean cholesterol value in healthyindividuals who are not suffering from a coronary artery disease or whenthe α-tocopherol/cholesterol value in the VLDL of the subject is lowerthan a mean α-tocopherol/cholesterol value in the healthy individualswho are no suffering from a coronary artery disease, the subject isjudged to have the risk of a coronary artery disease, and when theα-tocopherol/cholesterol value in the VLDL of the subject is not lowerthan the mean α-tocopherol/cholesterol value of the healthy individuals,the subject is judged not to have the risk of a coronary artery disease.5. A method of judging as to whether a subject is suffering fromdiabetes using a γ-tocopherol/α-tocopherol ratio measured in bloodcomprising the steps of: obtaining a blood sample from said subject,measuring the γ-tocopherol and α-tocopherol values contained in saidblood sample, and then calculating the γ-tocopherol/α-tocopherol ratioin the blood sample by dividing said measured γ-tocopherol value by saidmeasured α-tocopherol value, wherein when the γ-tocopherol/α-tocopherolvalue in the blood sample of the subject is lower than a meanγ-tocopherol/α-tocopherol value in healthy individuals who are notsuffering from diabetes, the subject is judged to suffer from diabetes,and when it is not lower than the mean γ-tocopherol/α-tocopherol valueof the healthy individuals, the subject is judged to be not sufferingfrom diabetes.